The long term objectives of the principal investigator's laboratory are to extend our understanding of the pathogenesis of acute pro-myelocytic leukemia (APL), and of the mechanisms by which the vitamin A derivative all-trans retinoic acid (ATRA) contributes to the cure of this disease. The specific theme of this proposal is the identification and functional characterization of genes involved in APL, and of genes regulated by ATRA in APL cells and PML-RARalpha-expressing cell lines. PML-RARalpha is the fusion gene, created by the 15;17 translocation that is felt to cause APL in humans (and exhibits leukemogenic activity in transgenic mice). In specific aim 1, a cDNA substraction technique will be used to identify genes that are downstream targets of PML-RARalpha. Candidate cDNAs will be evaluated for differential expression in APL and other myeloid leukemias, and assayed for their responsiveness to ATRA. Those which appear most likely to be involved in APL pathogenesis will be cloned and further characterized. In addition, cDNA microarray technology will be used to define a gene expression profile in APL cells and in PML-RARalpha-positive cell lines. In specific aim 2, a novel gene isolated from PML-RARalpha-expressing cells by subtractive hybridization, termed ARA-1 (ATRA-Regulated in APL), will be completely characterized. Since ARA-1 appears to be induced by ATRA specifically in PML-RARalpha-positive cells, the hypothesis that ARA- 1 is involved in the mechanism by which ATRA differentiates, and ultimately kills, APL cells, will be tested. Finally, in specific aim 3, the response of PML-RARalpha-expressing hematopoietic cells to ATRA will be further characterized. PML-RARalpha sensitizes TF1 erythroleukemia cells to ATRA-induced differentiation and apoptosis, and the magnitude of this effect is dependent on PML-RARalpha isoform type. These studies will be extended to identify regions within the PML-RARalpha protein which mediate this pro-differentiative and pro- apoptotic effect, and to further characterize the mechanism by which PML- RARalpha, in conjunction with ATRA, leads to differentiation and death of these cells. It is hoped that the research proposed here will lead to a more complete understanding of the pathogenesis of APL, and to strategies for transition of retinoid-based differentiation therapy from the laboratory to the bedside.